Radiation therapy is commonly used to treat various forms of cancers, either alone or in combination with chemotherapeutic agents. However, the effectiveness of radiation therapy varies depending on the nature of the cancer, the individual patient and whether radiation is used in combination with other treatments. Lokeshwar et al. (1995) Anticancer Res. 15(1):93-98. For example, Hennequin et al. (1996) Cancer Res. 56(8):1842-50 have shown that chemical agents such as paclitaxel (Taxol) or docetaxel (Taxotere) can either reduce or enhance radiation sensitivity of cancer cell lines depending on the drug concentration. Edelstein et al. (1996) Semin. Oncol. 23(2 Suppl. 5):41-7 report that the chemotherapeutic agent vinorelbine can be used to potentiate the antitumor effects of radiation in cycling cells. Where cancers develop resistance to chemotherapeutic agents, Siler et al. (1996) Cancer 77(9):1850-1853 report that successful clinical outcomes may be obtained by combining chemotherapy with external beam radiation. Thus, it would be useful to have a fast, reliable in vitro assay which could accurately predict an individual's response to radiation treatment and the effect of radiation in combination with other chemotherapeutic or radiation-sensitizing agents.
In cervical cancer, for instance, the majority of patients are diagnosed with early stage disease. Among 13,458 staged patients with cervical carcinoma registered by the Surveillance, Epidemiology and End Results (SEER) program between 1973 and 1987, 71% were diagnosed with the International Federation of Gynecology and Obstetrics (FIGO) stage I-IIA tumors. However, patients with more advanced lesions accounted for the majority of cervical cancer deaths during the same time period (Kosary (1994) Surgical Oncology, 10:31-46). These deaths occurred despite current radiotherapy protocols, often as a direct result of clinical treatment failure. Indeed, the 1973 and 1978 Patterns of Care Studies reported the 4-year in-field treatment failure rates to range from 20% in women with stage IIB cancers to 47% in those with stage IIIB lesions. In these studies, FIGO stage and laterality of disease were the only significant pre-treatment predictors of in-field control and survival (Lanciano et al. (1991) International J. Radiation Oncology, 20:667-76). The most current FIGO staging for cervical cancer is shown in Table 1.
TABLE 1 ______________________________________ Stage Description/Features ______________________________________ 0 Carcinoma in situ, intraepithelial carcinoma (cases of stage 0 should not be included in any therapeutic statistics for invasive carcinoma) I Carcinoma strictly confined to the cervix; extension to the corpus should be disregarded a. Preclinical carcinomas of the cervix; that is, those diagnosed only by microscopy Ia1. Minimal microscopically evident stromal invasion Ia2. Lesions detected microscopically that can be measured; the upper limit of the measurement should not show a depth of invasion of more than 5 mm taken from the base of the epithelium, either surface or glandular, from which it originates, and a second dimension, the horizontal spread must not exceed 7 mm; larger lesions should be staged as Ib Ib. Lesions of greater dimension than stage Ia2, whether seen clinically or not; space involvement should not alter the staging, but should be specifically recorded so as to determine whether it should affect treatment decisions in the future II Carcinoma extending beyond the cervix, but not onto the pelvic wall; involves the vagina, but not the lower one-third a. No obvious parametrial involvement b. Obvious parametrial involvement III Carcinoma extending onto the pelvic wall; (on rectal examination, there is no cancer-free space between the tumor and the pelvic wall; the tumor involves the lower one-third of the vagina; all cases with a hydronephrosis or nonfunctioning kidney) a. No extension onto the pelvic wall b. Extension onto the pelvic wall; urinary obstruction of one or both ureters on intravenous pyelogram (IVP) without the other criteria for stage III disease IV Carcinoma extending beyond the true pelvis or clinically involving the mucosa of bladder or rectum (a bullous edema, as such, does not permit a case to be allotted to stage IV) a. Spread to adjacent organs b. Spread to distant organs ______________________________________
Presumably, control of tumors in the pelvic region in patients with advanced cervical cancer depends not only on stage and tumor volume but also on intrinsic biologic radiation sensitivity. Studies assessing the intrinsic radiation sensitivity of various cancer cell lines are known in the art. Ruka et al. (1996) J. Surg. Oncol. 61(4):290-294 report that human soft tissue sarcoma cell lines do not show unusual radiation resistance when compared to human breast carcinomas or glioblastoma cell lines. Ma et al. (1996) Cell Biol Int. 20(4):289-292 describe how human diploid skin fibroblast cells exhibit heterogeneity in their response to radiation. None of these studies, however, indicate how in vitro radiation sensitivity could be used to predict clinical outcome.
To date, several trials investigating the utility of concomitant chemotherapy to improve radiation sensitivity have been inconclusive or have indicated that in vitro sensitivity is not predictive of clinical outcome. Ramsay et al. (1994) Int. J Radiation Oncology, 31(2):339-344 describe how a tetrazolium-based colorimetric assay (MTT) is not a useful predictor of radiosensitivity of lymphocytes derived from breast cancer patients. Similarly, Taghian et al. (1995) Int. J Radiat. OncoL Biol. Phys. 32(1):99-104, report that in vitro radiation sensitivity of human glioblastoma, squamous cell carcinoma, soft tissue sarcoma and cancer colon samples does not correlate with either in vivo radiation sensitivity or clinical outcome.
Immunohistochemical in vitro assays have also shown no correlation between the expression of genes involved in cancer and radiation response. Zaffaroni et al. (1995) Stem Cells 13:77-85 report that p53 expression does not correlate with in vitro response to gamma irradiation in primary cultures of human ovarian cancers and cutaneous melanomas. Bristow et al. (1996) Int. J. Radiat. Oncol. Biol. Phys. 34(2):341-355 also found no relationship between radiation resistance and metastatic potential in cells transfected with the p53 gene.
Another assay described by Griffon et al. (1995) European J. Cancer 31A(1):85-91, uses a multicellular tumor spheroid (MTS) three-dimensional model to determine radiosensitivity. MTS cultures exhibit characteristic phenotypes, including having a spheroid, three-dimensional shape. Griffon measured the doubling time and DNA ploidy of MTS in response to radiation treatments. Notably, this study does not indicate if radiosensitivity in vitro is predictive of clinical outcome. In addition, the assay described by Griffon is both labor and time intensive. Although established tumor cell lines often produce MTS, primary tumor specimens do so only occasionally. Furthermore, the specimens obtained from patients must be grown for 4 to 5 days to develop spheroids which can be exposed to radiation. After radiation, the MTS must be cultured for at least 7 days before radiosensitivity can be measured. Thus, even if an MTS culture can be established from a patient sample, radiation response results are not available for a minimum of 11 days.
Chromosomal painting methods have also been used to try and predict radiation sensitivity. Dunst et al. (1995) Strahlenther Onkol. 171(10):581-586 describe how patients with abnormal and extreme radiosensitivity could possibly be identified by in-vitro testing of lymphocytes. Radiation sensitivity was determined by the amount of chromosomal damage, as measured by fluorescence in-situ hybridization (FISH). The assay described in Dunst is not predictive of clinical outcome in patients having decreased or normal radiosensitivity.
Clonogenic proliferation inhibition assays on fresh tumor explants have been the mainstay of in vitro radiation and chemotherapy response determination and have demonstrated prognostic value in human solid tumor models. C. M. West et al. (1993) British J. Cancer, 68:819-23 recently reported that patients with cervical carcinoma treated with radiotherapy alone and followed for a minimum of two years showed a correlation between in vitro response and clinical outcome. Life table analysis demonstrated a significantly longer disease-free survival in patients whose tumors were found to have higher than average in vitro response to radiation exposure. However, clonogenic assays have not been routinely incorporated in clinical trials due to several disadvantages, such as labor intensiveness, high cost, poor standardization due to low plating efficiencies and clump artifact, and long assay duration.
With the advent of in vitro .sup.3 H-Thymidine incorporation assays, several limitations of clonogenic techniques have been overcome. Specifically, .sup.3 H-Thymidine incorporation assays eliminate clump artifact, render reliable results in 85% of tumor explants examined, and can be completed within 6 days. While a variety of in vitro endpoints have been associated with radiation and chemotherapeutic drug response, proliferation inhibition assays have demonstrated the greatest clinical utility. This utility is based principally upon their high degree of accuracy at predicting resistance to chemotherapy. One such in vitro drug response assay, the Extreme Drug Resistance (EDR) assay, is currently being investigated by the Gynecologic Oncology Group (GOG) for its ability to predict clinical drug resistance and disease-free survival in patients undergoing primary chemotherapy for epithelial ovarian cancer. (see, Manetta et al. (ongoing) Protocol GOG #118). The EDR assay is a proliferation inhibition assay based on .sup.3 H-Thymidine incorporation by tumor cells grown in soft agar. In all solid tumor models studied thus far, results of the EDR assay have correlated with those of the clonogenic stem cell assay (Freuhauf et al. (1993) in CANCER: PRINCIPLES & PRACTICE OF ONCOLOGY, eds. De Vita et al. suppl. 7:12).
There remains a need, however, for an assay which could confidently predict radiation resistance. The assay could predict local treatment failure with such combination regimens and could identify patients for whom unnecessary toxicity could be prevented and alternative therapies considered. Applicants describe herein a novel assay that can predict radiosensitivity of cancer cells, clinical resistance to primary radiation, interactions between chemotherapy and radiation, and clinical outcome.